This invention relates to a novel separation method for separating a target substance (metal ions, drugs, biological components, etc.) with the use of a separatory material wherein the interaction force of a substance (ligand) interacting with the target substance can be reversibly changed in an aqueous system due to a structural change or a polarity change of a stimulus-responsive polymer under a physical stimulus, and a separatory material such as a packing to be used in this method.
The most effective and efficient means employed at present for separating and purifying biological components and drugs include ion exchange chromatography, reversed phase chromatography, affinity chromatography, etc. In recent years, bioengineering procedures have made remarkable advances and physiologically active substances (recombinant proteins, glycoproteins, etc.) are produced thereby on a mass scale. Under these circumstances, there is a growing requirement for methods by which these products can be quickly and efficiently separated and purified without inactivation.
In the chromatographic techniques as cited above, however, target substances (biological components, drugs, recombinant proteins, glycoproteins, etc.) are eluted by changing the salt concentration, organic solvent concentration, pH value, etc. of eluents. It is known that, in such a case, the pH value, organic solvent, etc. frequently bring about severe conditions for the target substance and thus lower the recovery yield thereof. In addition, the salt, organic solvent, pH-regulating agent, etc. employed in eluting the target substance should be finally eliminated by desalting, drying, etc. Therefore, an additional step is required, after the completion of the separation and purification of the target substance, to perform an operation for eliminating the salt, organic solvent, pH-regulating agent, etc. As a result, the activity and yield of the final product are often lowered.
When the target substance is eluted by a chemical means with the use of a salt, an organic solvent, a pH-regulating agent, etc., the chemical (i.e., the salt, organic solvent, pH-regulating agent, etc.) contained in the eluent causes the above-mentioned problems of inactivation, a decrease in the yield, etc. It is expected that if a physical change induced by, for example, heat, light or a magnet could affect the elution of a target substance, the target substance would be eluted not by a chemical means but by a physical one, thus solving the problems of inactivation, a decrease in the yield, etc.
Recently, separation material comprising stimulus-responsive polymers covalently attached to ion exchanging groups have been described. See for instance JP application 140722/98 and corresponding WO application WO99/61904.
Galaev et al (J. Chromatog. A 684 (1994) 37-43 describe temperature elution of lactate dehydrogenase (LDH) in a chromatographic system in which a dye (ligand) is covalently attached to a base matrix which also carries a physically adsorbed temperature responsive polymer.
Hofman et al., (WO 8706152) describes a separation method in which the ligand is attached to a temperature responsive polymer. Binding and elution of the target substance occur at the same side of the critical solution temperature.
There are also a number of publications describing chromatography based on separatory material comprising stimulus-responsive polymers but without having a ligand covalently attached to the temperature-responsive polymer. Gewehr et al (Macromolecular Chemistry and Physics 193 (1992) 249-256) describes gel chromatography on porous silica beads coated with a temperature-responsive polymer. Hosoya et al (Anal. Chem. 67 (1995) 1907-1911); Yamamoto et al. (Proc. 114th National Meeting of the Pharmaceutical Society of Japan, Tokyo (1994) 160; Kanazawa et al (Yakugaku Zasshi 117 (10-11) (1997) 817-824; Kanazawa et al (Anal. Chem. 68(1) (1996) 100-105); Kanazawa et al (Anal. Chem. 69(5) (1997) 823-830); Kanazawa et al (J. Pharm. Biomed. Anal. 15 (1997) 1545-1550); Yakushiji et al (Langmuir 14(16) 1998) 4657-466268); Kanazawa et al (Trends Anal. Biochem. 17(7) (1998) 435-440); Yakushiji et al (Anal. Chem. 71(6) 1999) 1125-1130); Grace and Co (EP 534016); Okano (JP 6-108643) describe reversed phase chromatography on matrices covered by a thermoresponsive olymer for the separation of biomolecules. The matrices may be porous. The hydrophobic groups utilized are inherent in the polymer as such. There is no ligand that has been covalently attached to the polymer after polymerisation.
From this point of view, the present inventors have conducted intensive studies and developments on the elution of a target substance by a physical means to thereby solve the above problems. As a result, they synthesized a composite material comprising a stimulus-responsive polymer with a ligand molecule by binding poly(N-isopropylacrylamide) to a molecule (i.e., a ligand molecule) capable of interacting with a target substance. Subsequently they have found out that use of this composite material makes it possible to obtain a separatory material which undergoes a change in the interaction between the ligand molecule and the target substance under a physical stimulus. The present invention has been completed based on this finding.
The present invention relates to a method for separating substances characterized in that a composite material comprising a stimulus-responsive polymer and a substance (ligand) interacting with a target substance undergoes a physical or chemical change of the stimulus-responsive polymer under a physical stimulus so that the environment of the interaction between the target substance and the molecule interacting therewith is physically or chemically changed. This means that the target substance can be released from the ligand and also from the separatory material, thus effecting separation of the target substance from the composite material or separatory material.
The present invention further relates to a method for separating substances characterized by comprising (a) binding a target substance on a stationary phase of a separatory material (including chromatographic packing) chemically modified with a composite material comprising a stimulus-responsive polymer and a substance (ligand) interacting specifically with the target substance; then (b) changing continuously or stepwise the temperature, preferably by external means, to thereby weaken the interaction between the ligand and the target substance; and eluting the chromatographic packing by a mobile phase while maintaining a temperature which permit a weakened interaction between the ligand and the target substance thus effecting separation. The mobile phase may be a liquid, for instance aqueous.
The present invention further relates to a separatory material (for example, a chromatographic packing) wherein the ability of a substance to recognize a molecule can be changed due to a physical stimulus.
Another embodiment of the invention is a method for the separation of one or more target substances from a liquid. This embodiment comprises the steps of substances from
(a) bringing a liquid sample (I) containing a target substance in contact with a separation medium/separatory material (including a chromatographic packing) which is functionalized with a ligand which is capable of binding to the target substance, said contact being under conditions permitting binding of said target substance to said ligand;
(b) contacting said carrier with a liquid (II) not containing said at least one target substance under conditions such that the target substance is released from said ligand to liquid (II).
Between steps (a) and (b) the liquid sample is preferably separated from the separatory material which in turn may be washed before step (b). After step (b), liquid (II) may be separated from the separatory material. The target substance, if so desired, may be worked up from liquid II.
With respect to target substances in form of biological molecules such as those having nucleotide structure (including nucleic acids), polypeptide structure (including proteins), carbohydrate structure, steroid structure etc the liquids used typically have been aqueous. This embodiment of the invention is characterized in that
(i) said separatory material comprises a stimulus-responsive polymer as defined elsewhere in this specification, which polymer has been functionalized with the ligand, preferably by covalent attachment of the ligand after the polymer has been formed, and
(ii) subjecting in step (a) and at least during binding of the target substance to the ligand, the separatory material to a stimulus at a level/intensity at which the stimulus-responsive polymer in a conformation enhancing binding of the target substance to the ligand, and
(iii) subjecting in step (b) and at least during release of the target substance from the ligand the separatory material to a stimulus at a level/intensity at which the stimulus-responsive polymer is in a conformation hindering binding of the target substance to the ligand.
The level/intensity of the stimulus is on opposite sides of the critical level/intensity for the stimulus-sensitive polymer used and other conditions applied in the respective step. The process can be made cyclic in case step a is repeated after step b, typically after extra washing/regeneration steps and equilibration steps.
Various embodiments of the inventive method may be carried out in a batch-wise or a chromatographic mode. Chromatographic modes, for instance, may be carried out by permitting the various liquids in plug flow (mobile phase) to pass through a bed of the separatory material while subjecting the bed to the appropriate stimulus for the individual steps and stimulus-responsive polymer used. The bed may be a porous monolith or a bed of packed or fluidised particles. Batch-wise modes in particular concerns suspended particles in combination with turbulent flow and/or liquids.